ORAI1 and ORAI2 modulate murine neutrophil calcium signaling, cellular activation, and host defense Derayvia Grimesa,1, Ryan Johnsona,1, Madeline Pashosa, Celeste Cummingsa, Chen Kangb, Georgia R. Sampedroa, Eric Tycksenc, Helen J. McBrided, Rajan Sahb, Clifford A. Lowelle, and Regina A. Clemensa,2 aDepartment of Pediatrics, Washington University School of Medicine, St. Louis, MO 63110; bDepartment of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110; cMcDonnell Genome Institute, Washington University School of Medicine, St. Louis, MO 63110; dInflammation Research, Amgen, Thousand Oaks, CA 91320; and eDepartment of Laboratory Medicine, University of California, San Francisco, CA 94143 Edited by Michael D. Cahalan, University of California, Irvine, CA, and approved August 11, 2020 (received for review May 5, 2020) Calcium signals are initiated in immune cells by the process of in isoform features, such as activation or inactivation kinetics and store-operated calcium entry (SOCE), where receptor activation sensitivity to modulatory factors, also influence CRAC-channel triggers transient calcium release from the endoplasmic reticulum, function (2–10). ORAI1 and ORAI2 are broadly expressed in im- followed by opening of plasma-membrane calcium-release acti- mune cells, and humans with ORAI1 mutations develop a severe vated calcium (CRAC) channels. ORAI1, ORAI2, and ORAI3 are known combined immunodeficiency-like immunodeficiency, highlighting to comprise the CRAC channel; however, the contributions of indi- the importance of this isoform in immune function (11, 12). In mice, vidual isoforms to neutrophil function are not well understood. ORAI1 also appears to be the dominant functional isoform in im- Here, we show that loss of ORAI1 partially decreases calcium influx, mune cells, with substantial deficits in ORAI1-deficient T cells, while loss of both ORAI1 and ORAI2 completely abolishes SOCE. In mast cells, and platelet SOCE (11, 13, 14). The role of ORAI2 is other immune-cell types, loss of ORAI2 enhances SOCE. In contrast, less clear; however, a recent report by Vaeth et al. (15) demon- we find that ORAI2-deficient neutrophils display decreased calcium strated that in naïve CD4+ T cells, dendritic cells, and macro- influx, which is correlated with measurable differences in the regu- phages, ORAI2 appears to play a modulatory role, such that lation of neutrophil membrane potential via KCa3.1. Decreased SOCE in ORAI1-, ORAI2-, and ORAI1/2-deficient neutrophils impairs deletion of ORAI2 enhances SOCE. Mice with deletion of ORAI2 multiple neutrophil functions, including phagocytosis, degranula- combined with T cell-specific deletion of ORAI1 are protected in tion, leukotriene, and reactive oxygen species (ROS) production, models of graft vs. host disease, but are more susceptible to viral IMMUNOLOGY AND INFLAMMATION rendering ORAI1/2-deficient mice highly susceptible to staphylococ- infection, demonstrating the relevance of this signaling pathway in T cal infection. This study demonstrates that ORAI1 and ORAI2 are the cell-mediated immune responses (15). primary components of the neutrophil CRAC channel and identifies Calcium influx mediates critical neutrophil functions, such as subpopulations of neutrophils where cell-membrane potential func- phagocytosis, degranulation, reactive oxygen species (ROS) tions as a rheostat to modulate the SOCE response. These findings production, and cytokine synthesis (16–26), but less is known have implications for mechanisms that modulate neutrophil func- about the molecular machinery that drives SOCE in neutrophils. tion during infection, acute and chronic inflammatory conditions, Previous work from our group and others demonstrated that and cancer. Significance neutrophil | membrane potential | calcium signaling | store-operated calcium entry | ORAI1 Neutrophils are essential for host defense against pathogens. Although calcium signals direct numerous cell functions, the eutrophils are increasingly appreciated to display a diverse molecular machinery that orchestrates calcium influx into Narray of phenotypes and behaviors; however, the signaling neutrophils is poorly defined. We demonstrate that ORAI1 and pathways that govern neutrophil responses are incompletely ORAI2 are key components of the mouse neutrophil calcium understood. As a central signaling mediator, cytoplasmic calcium release-activated calcium channel and are essential for neu- concentrations are tightly controlled, and, in resting cells, cal- trophil bactericidal function. We find that neutrophils separate cium is sequestered outside the cell or within organelles such as into two populations with distinct regulation of the membrane the endoplasmic reticulum (ER). Calcium entry into neutrophils potential during calcium influx, which influences the magni- occurs primarily by store-operated calcium entry (SOCE), tude of SOCE and the effect of ORAI isoform deficiency. These whereby receptor activation initiates a signaling cascade, culmi- findings advance understanding of the mechanics of neutrophil nating in activation of phospholipases, which liberate diac- calcium signaling, identify neutrophil subpopulations where the cell-membrane potential modulates the calcium response, ylglycerol and inositol triphosphate (IP3) from membrane-bound and suggest mechanisms that regulate neutrophil function phosphatidylinositol 4,5-bisphosphate (1). IP3 binding to the ER during infection and inflammation. IP3 receptor releases “store” calcium from the ER, resulting in transient elevation of cytoplasmic calcium. Stromal interaction Author contributions: C.K., G.R.S., R.S., C.A.L., and R.A.C. designed research; D.G., R.J., molecule (STIM) calcium sensors in the ER membrane undergo M.P., C.C., C.K., G.R.S., and R.A.C. performed research; E.T., H.J.M., and R.S. contributed oligomerization and conformational change triggered by ER new reagents/analytic tools; C.K., E.T., and R.A.C. analyzed data; and R.A.C. wrote calcium depletion and gate calcium-release activated calcium the paper. (CRAC) channels in the plasma membrane. Opening of these Competing interest statement: H.J.M. was an employee and shareholder of Amgen, Inc. at channels allows sustained calcium entry into the cell and acti- the time the studies were performed. vation of calcium-dependent cellular processes. This article is a PNAS Direct Submission. The CRAC channel is a hexamer comprising varying ratios of Published under the PNAS license. three ORAI family members, ORAI1, ORAI2, and ORAI3. When 1D.G. and R.J. contributed equally to this work. overexpressed with STIM1, all three isoforms display features 2To whom correspondence may be addressed. Email: [email protected]. characteristic of CRAC channels, signified by an inwardly rectifying, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ highly calcium-selective, low-conductance current and fast calcium- doi:10.1073/pnas.2008032117/-/DCSupplemental. dependent inactivation (2, 3). Expression patterns and differences www.pnas.org/cgi/doi/10.1073/pnas.2008032117 PNAS Latest Articles | 1of12 Downloaded by guest on September 26, 2021 STIM1 and STIM2 cooperatively regulate neutrophil SOCE; (as measured by qPCR) and no significant compensatory alter- however, the role of different ORAI channel components is less ation in expression of other ORAI isoforms (Fig. 1D). ORAI clear (19, 27). While knockdown of ORAI1 in neutrophil-like cell protein largely recapitulated mRNA levels; however, ORAI2 lines impairs SOCE, results from primary human and mouse protein was decreased in ORAI1-deficient neutrophils, whole neutrophils have been mixed, with either moderately decreased or splenocytes, and naïve CD4+ T cells, suggesting that ORAI2 normal SOCE observed in ORAI1-deficient neutrophils (24, stability is potentially altered in the absence of ORAI1 (Fig. 1 E VavCre VavCre 28–32). The reason for these discrepancies is unclear; however, and F and SI Appendix, Fig. S2 B and C). Orai1 , Orai2 , VavCre across all studies, loss of ORAI1 at most only partially impairs and young Orai1/2 mice had normal numbers of circulating SOCE, implying that other channel components, such as ORAI2 neutrophils with no changes in surface phenotype (SI Appendix, VavCre or ORAI3, also contribute to SOCE in neutrophils. Fig. S2 D–F). Orai1/2 mice are known to have decreased Here, we show that ORAI1 and ORAI2 are the predominant regulatory T cells and develop lymphoproliferation with age (15). ORAI isoforms expressed in mouse neutrophils and that their We found that circulating neutrophil numbers were increased in expression is differentially regulated by inflammatory stimuli. mice >10 wk old; therefore, we used 6-wk-old mice, which had Using mice with hematopoietic deletion of Orai1 and/or Orai2,we no appreciable difference in resting neutrophil number or sur- show that SOCE is diminished in the absence of ORAI1 and face phenotype (SI Appendix, Fig. S2 D–F). completely abolished in double-mutant cells. In contrast to other immune-cell types, deletion of Orai2 impairs, rather than aug- ORAI1 and ORAI2 Cooperatively Regulate SOCE in Neutrophils. To ments, SOCE in mouse neutrophils. In investigating the mecha- analyze the contribution of ORAI1 and ORAI2 to neutrophil VavCre VavCre nism underlying this neutrophil-specific phenotype, we observed SOCE, we measured calcium influx in Orai1 , Orai2 , VavCre that bone marrow neutrophils display heterogeneous calcium re- and Orai1/2 neutrophils. ER store release